Activities of Anionic and Cationic Trypsins in the Temperature Range from 5 to 37°C. Mutant Anionic Trypsins as a Model of Cold-Adapted (Psychrophilic) Enzymes

A. G. Mikhailova^1*, L. D. Rumsh¹, M. Dalgalarrondo², J.-M. Chobert², and T. Haertle²

¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia; fax: (095) 335-7103; E-mail: anna@enzyme.siobc.ras.ru

²Institut National de la Recherche Agronomique, B. P. 71627, 44316 Nantes Cedex 3, France; fax: 332-40-67-50-84; E-mail: haertle@nantes.inra.fr

^* To whom correspondence should be addressed.

Received November 22, 2002

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Temperature dependences of kinetic constants (k_cat and K_m) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of cold-adapted fishes. The anionic and cationic trypsins were found to differ in organization of the S₁-substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S₁) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K_m value) to the anionic and cationic trypsins and on the catalytic activity k_cat should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.

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KEY WORDS: anionic and cationic trypsins, psychrophilic trypsins, mutant trypsins, temperature dependence

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